Perfusion, Cryosectioning, and Immunofluorescence of Mouse Spinal Cord

After terminal anesthesia by barbiturate overdose, mice were perfused transcardially with a phosphate buffered saline rinse followed by either 4% paraformaldehyde or 10% formalin. Spinal cords were removed, post-fixed overnight, and cryoprotected in buffered 30% sucrose for 48 hours. Frozen sections (30 μm) were prepared using a cryostat microtome (Leica) and processed for immunofluorescence as described7 (link)12 (link)13 (link). Primary antibodies were: rabbit anti-GFAP (1:1000; Dako, Carpinteria, CA); rat anti-GFAP (1:1000, Zymed Laboratories); sheep anti-BrdU (1:300, Maine Biotechnology Services, Portland, ME); rat anti-CD133 (1:200; Millipore, Temecula, CA); and rat anti-vimentin (clone # 280618; 1:150; Novus Biologicals, Littleton, CO). Fluorescence secondary antibodies were conjugated to: Alexa 488 (green) or Alexa 405 (blue) or to Cy3 (550, red) or Cy5 (649, far red) all from Jackson Immunoresearch Laboratories. Nuclear stain: 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI; 2 ng/ml; ThermoFisher). Sections were coverslipped using ProLong Gold anti-fade reagent (InVitrogen, Grand Island, NY). Sections were examined and photographed using deconvolution fluorescence microscopy and scanning confocal laser microscopy (Zeiss, Oberkochen, Germany).